(a) Field of the Invention
The present invention relates to a pharmaceutical composition for protecting neurons or for preventing and treating ischemic neuronal diseases, more precisely a pharmaceutical composition for protecting neurons or for preventing and treating ischemic neuronal diseases comprising a plant extract or a single compound extracted therefrom which is effectively used for the treatment of degenerative brain diseases caused by neuronal apoptosis as an effective ingredient and functional health food comprising the same.
(b) Description of the Related Art
Nerve system is largely composed of neurons and neuroglia cells, and the neuroglia cells take 90% of the total brain cells in number and take 50% of the total brain in volume. The neuroglia cells in central nervous system are composed of astrocytes, oligodendrocytes and microglia. And microglia cells are inflammatory cells that take 5-10% of the total brain cells and have been confirmed to be macrophage like immuno-functional cells which are activated when brain damage, including degenerative brain disease and stroke, occurs by some reasons even though their role in brain under normal conditions has not been disclosed yet (Minghetti L, et al., Prog. Neurobio. I. 54(1), pp 99-125, 1998). Unlike normal cells, the activated microglia cells exhibit active phagocytosis and vigorous proliferation, so that they induce expressions of various genes such as cytokine, inducible nitric oxide synthase (iNOS) and cyclooxygenase-s (COX-2), suggesting that they generate various inflammation mediators (Dawson V L, et al., J. Neurosci., 13, pp 2651-2661, 1993). There have been so many proofs reported saying that such inflammation mediators generated as the above are one of the reasons that cause degenerative brain diseases such as Alzheimer's disease, Parkinson's disease, stroke and dementia (wood P L, et al., Neurol. Res., 17, pp 242-248, 1995).
The inflammation mediators generated by the activated microglia cells produce nitric oxide (NO) (Feldman P L, et al., Chem Eng. News., 12, pp 26-38, 1993). Nitric oxide is generated by nitric oxide synthase (NOS) and this enzyme can be classified into constitutive NOS (cHOS) that produces nitric oxide for normal physiological functions including neurotransmission, blood clotting and blood pressure control; and inducible nitric oxide that is specifically induced under special circumstances. Generation of constitutive NOS (cNOS) depends on calcium (Ca2+) and calmodulin (CaM) and only small amount of cNOS can be generated within a short period of time by a certain stimulus. On the other hand, the generation of inducible NOS (iNOS) is quite independent and iNOS is not activated under the normal conditions but once its expression is induced by the stimulus of lipopolysaccharides, iNOS can be mass-produced for a certain period of time and thus iNOS produces inflammatory cytokines such as interferon-gamma (INF-gamma), interleukin-1beta, cancer necrosis factor-alpha, etc. (Mayer B., et al., FEBS Lett, 288, pp187-191, 1991). Excessive nitric oxide generated by inducible NOS and inflammatory cytokine generated aftermath lower blood pressure significantly by extending blood vessels in sepsis patients, make inflammation worse by accelerating biosynthesis of inflammation mediators such as prostaglandins by activating COX-2, and is deeply involved in the development and progress of brain diseases resulted from neuronal apoptosis and aggravated inflammation.
It has been an urgent request therefore to develop a safe novel neuroprotective agent with fewer side effects that can inhibit significantly the expressions of inflammatory cytokines causing brain disease and making inflammation worse. And it is also requested to establish a specialized medicine market for neuroprotective agents.
Acetylshikonin used in this invention is a naphtoquinone compound extracted and isolated from Lithospermum erythrorhizon Sieb. Et Zucc, an oriental herb medicine which has long been used for the treatment of dermatitis in Korea and China.
Lithospermum erythrorhizon Sieb. Et Zucc (gromwell) is a perennial plant. Its root contains acetylshikonin, shikonin, alkannan, isobutyrylshikonin, beta/beta-dimethylacrylshikonin, beta-hydroxyisovalerylshikonin, and teracryishikonin, and the purple color of the root is attributed to acetylshikonin. Shikonin or acetylshikonin has functions of inhibiting edema, promoting granulation growth and accelerating wound healing, in particular boosting regeneration of destroyed or damaged cells by burn or ulcer. These compounds also have antimicrobial activity against bacteria or virus and exhibit a strong capillary permeability. In addition, these compounds have been known to have functions of improving blood circulation and reducing blood sugar and anticancer activity as well (Jung B S and Shin M K, Hyangyak daesajeon, 1998, pp 891-892). However, there has been no reports in association with the therapeutic effect of the extract of Lithospermum erythrorhizon Sieb. Et Zucc or acetylshikonin on brain disease by inhibiting inflammatory cytokine and preventing neuronal apoptosis.
Therefore, the present inventors investigated in this invention the effect of the extract of Lithospermum erythrorhizon Sieb. Et Zucc and acetylshikonin separated therefrom on inflammatory cytokine by various biochemical experiments, examined the possibility of interruption of nitric oxide and inflammatory cytokine by molecular biological approaches and further investigated the mechanism of the extract of Lithospermum erythrorhizon Sieb. Et Zucc and acetylshikonin for protecting neurons particularly from ischemic neuronal apoptosis by using animal models with local brain ischemia.
As a result, the extract of Lithospermum erythrorhizon Sieb. Et Zucc of the present invention extracted by water, low-alcohol or their mixed solution and acetylshikonin separated and isolated from the extract inhibited expressions of various inflammatory cytokines including inducible nitric oxide synthase in vitro, and thereby the inventors confirmed the mechanism of the extract and acetylshikonin to interrupt over-production of nitric oxide and to protect neurons from apoptosis caused by brain ischemia by using rats with local brain ischemia induced by middle cerebral arterial occlusion. In conclusion, the present inventors completed this invention by confirming that acetylshikonin has an activity of protecting neurons and a function of inhibiting neuronal apoptosis by investigating the therapeutic effect and therapeutic mechanism of acetylshikonin for ischemic brain diseases.